The Small-Molecule Compound Baicalein Alleviates Experimental Autoimmune Encephalomyelitis By Suppressing Pathogenetic CXCR6~+CD4 Cells

Objective:CXC chemokine receptor 6(CXCR6)-based immunotherapy plays a significant role in autoimmune diseases;However,there have been no reports to date on small-moleculemediated suppression of pathogenic CXCR6+Th cells for the treatment of autoimmune diseases.Baicalein,a flavonoid isolated from Scutellarin baicalensis(Huang Qin),was shown to exert therapeutic effects on MS,but the underlying mechanisms are largely unknown.The purpose of this study is to reveal the mechanism of baicalein inhibiting CXCR6+CD4 cell-mediated treatment of autoimmune encephalomyelitis on the animal model of experimental autoimmune encephalomyelitis(EAE),so as to provide scientific basis for the clinical application of baicalein in the treatment of multiple sclerosis.Methods:1.In vitro study:The MTT method was used to measure the cytotoxicity of baicalein;Th17 cell differentiation test was used to detect the level of IL-17 secretion and the proportion of IL-17A by ELISA and flow cytometry respectively.2.In vivo study:the model of experimental autoimmune encephalomyelitis(EAE)was established by MOG35-55,and the therapeutic effect of baicalein was evaluated by Knoz score.HE staining and LFB staining were used to detect and evaluate the pathological injury of spinal cord tissue of EAE mice.Enzyme-linked immunosorbent assay(ELISA)was used to detect the production of cytokines during the specific immune response induced by MOG35-55.Elisa method was used to detect the levels of anti-MOG35-55 IgG,IgG3,IgG2a in serum.Quantitative polymerase chain reaction(qRT-PCR)was used to detect the expression of related genes in Thl and Th17 cells,including IL17a、Rorc and Ifng.Surface markers(CD3,CD4,CD8,CXCR6,CD44,CD62L,and CD11b),Treg cells,and intracellular cytokines(IL-17A and IFN-γ)were detected by flow cytometry.Results:1.In vitro experiments showed that baicalein did not produce drug toxicity at the concentration of 1-3 μM,and at this concentration,baicalein significantly inhibited the differentiation of initial T cells into Thl and Th17.2.Oral administration of baicalein(25 mg/kg)could significantly reduce the incidence and clinical score of EAE mice,reduce the degree of demyelination of EAE,and selectively block the production of IL-17A in the specific immune response induced by MOG35-55(P<0.05).The results of ELISA assay showed that in baicalein intervention group,the total antibody level of anti-MOG35-55(P<0.01)and IgG3 subtypes decreased significantly(P<0.05),while the production of IgG2a subtypes increased significantly(P<0.01).The results of qRT-PCR showed that Th17-related genes were significantly down-regulated in baicalein intervention group,and there was significant difference between IL17a and Rorc(P<0.05).In addition,the expression of CD4 cell effectors(CD44hi CD62Llow)and pathogenic Th17 cells was decreased by baicalein treatment.Furthermore,baicalein treatment largely decreased CXCR6+CD4 and CD8 cells and prominently inhibited CXCR6+Th17 cells in EAE.Conclusion:Baicalein is a safe and effective small molecular compound,which can improve the symptoms of multiple sclerosis,and its mechanism may be through the inhibition of pathogenic CXCR6+CD4 cells to improve EAE.